While Roundup is almost instantaneously adsorbed in soil with either clay or humic acid content, becoming unavailable to roots, that is apparently NOT the case in mulching materials. And roses commonly send roots up into the mulch. That's the reason many rosarians forbid the use of Roundup in the rose garden (we certainly forbid it at FSC). I notice that the symptoms reported occurred specifically in sprayed (as opposed to painted) treatments, so while they may have been shielded against horizontal drift, there is no reason to believe that there was not any run-off onto the mulch below. No mention was made of any attempt to rule out in-mulch spread via non-grafted roots. I must remember to explore that possibility with the folks at FPMS. But until we can be sure such precautions were taken, that is my theory of how symptoms could occur in plants a meter apart -- a distance roots would easily travel in an established plant.

There is one other confusing set of results in the Davis spread 2007 paper. They did 4 multiflora experiments with mixed infections of PNRSV and ApMV (2 with one source of mixed viruses and 2 with a different source for the mixed viruses.

For the 2 hedged experiments after 3 years, the "mixed virus source 1" experiment found one plant was positive for PNRSV, two plants were positive for ApMV, and seven plants were positive for both (10 plants tested negative). The "mixed virus source 2" experiment found two were positive for PNRSV and one was positive for both (20 plants tested negative).

For the 2 unhedged experiments after 3 years, the "mixed virus source 1" experiment found found nine plants were positive for PNRSV, two plants were positive for ApMV, and five plants were positive for both (11 plants tested negative). The "mixed virus source 2" experiment found ten were positive for PNRSV and two were positive for both (15 plants tested negative).

H. Kuska comment: If the viruses were uniformally distributed in the original infected plants, and if there were only root graph transmission; I would of expected only results where both viruses were detected equally.

The authors addressed the results in the paper. They said that the unequal distribution could of been the result of "differential spread, strain differences, or imperfect detection methods".

I will try to address these three possibilities.

Differential spread - I am not sure what is meant here. Do they mean that because of the "uneven distribution of viruses in roses" only the virus(s) that was/were at the part of the root that formed the graft were able to make the crossover? Or do they mean that the plants immune system was more efficient against ApMV than against PNRSV?

Strain differences - one or both the PNRSV and ApMV in the first source used could be more or less potent than the corresponding virus from the other source. Thus, only the same source data should be compared if this was a contributing factor.

Imperfect detection methods - ELISA testing was used so it is possible that some infections were below the detection limits.

------------------------------------
H. Kuska summary: from what is now known about unequal distribution of viruses in plants, I would "predict" that unequal distribution along with any differential differences in immune function would be factors - if root grafts prove to be the only source of spread. If sap spread (branches rubbing against each other) contributes then the differences in the thermal stability of the viruses in the sap may account for some or all of the observed differences.

Thank you.

The following was stated by malcolm_manners 9b C. Fla. (My Page) on Thu, Dec 31, 09 at 18:44: "But to claim that entire buds, or leaves, or branches, of a bush could be virus-free while other areas are infected, simply is not supportable by any published research, and we have major reasons to believe it not to be true."

H. Kuska comment: I have presented a number of published reviewed papers that report that they could not find virus in parts of a plant such as leaves and even in one paper, single node cuttings that were grown out for a long enough time so that the cuttings leafed out. Here is the single node cutting experimental (on a rose) details:

"ELISA test of cuttings. Six to eight single-node cuttings (approximately 5 cm long) of healthy and PNRSV-infected Anna and Lnidas plants were excised in the medial zone of floral stems at anthesis. Basal ends of the cuttings were dipped into 0.5% (wt/wt) ï¢-indole butyric acid. To protect them against fungal infection, they were treated with 800 mg of pyrimethanil (SCALA; AgrEvo, Berlin) per liter. Cuttings were cultivated in perlite in a heated glasshouse (minimal temperature 25°C) at 80% relative humidity. After 15 days, the rooted cuttings were transferred to a culture room at 22°C with a 16-h photoperiod. For each cutting, leaflets from the axillary leaf were tested by ELISA when cuttings were removed from the mother plant. Young shoots grown from the axillary buds of each cutting were tested by ELISA when they were 2 to 5 cm long."

Table 1 in the paper tells the number of days after the mother plants were tested that the leafed out cuttings were tested (30 to 86) and the number of samples (48 to 76).

In the conclusion section they state: "A total of 89% (59 out of 66) of Anna stems was entirely PNRSV negative when axillary shoots from these stems developed after the decapitation of the floral stem, after the development of cuttings, or grown in vitro were tested by ELISA. ELISA tests of leaves from these stems were all negative. Since IC-RT-PCR conducted in parallel on all the explants from three (out of seven) of these stems also were negative, we suspect that these stems were virus free, even if they belonged to infected plants, or that the viral titer was very low in the stems."

Please notice the conservative word "suspect" and the conservative statement "or that the viral titer was very low in the stems." In my experience this is the normal way of stating results as scientists realize that our interpretations and experimental methods are not normally "absolutely definitive".

Here is a link that might be useful: Moury et al year 2000 paper

You're arguing against yourself, Henry. Your last paragraph goes against everything you've been doing here for years -- the statement is true, but you always emphasize such scientific conservatism as proving your point, rather than taking them as the authors meant them. A case in point would be FPMS's caveats about the reliability of testing -- one needs such caveats if one is merely 99.999999999999% sure of something. Yet you emphasize those caveats as saying that what FPMS does is unreliable.

In this case, if they really are finding complete freedom from virus, then your arguments in this entire discussion are infinitely weaker than even I thought they were -- no sort of treatment at all would be necessary -- just take a bunch of cuttings of room-temperature-grown roses, and you can clean up your crop! There! We're done now. Mosaic is not a problem worth your ever mentioning again! Now, of course, neither of us believes that to be the case, but that is, in a sense, what you're claiming by emphasizing these results. On the other hand, while I generally do believe and agree with the last statement above, in this particular case, I very strongly suspect that it is true that it is a case of too-low titre, rather than complete absence of virus, particularly since this paper is now 10 years old, and still, no one is claiming to be able to make virus-free plants through simply taking cuttings.

It occurs to me that there is another possibility here: they pointed out that different strains of PNRSV are more or less pathogenic in Rosa, and that they used several strains in this research. Do we have any reason to think the strain being discussed here (the one they couldn't find in 'Anna') is one that we actually have in common rose mosaic in the USA (or France, for that matter)? I see no reason to think so from this paper. And as always, I'm not interested in discussing other viruses (or other strains of viruses) as if they were "rose mosaic disease." Notice that they state "We evaluated the detection method in cuttings and in vitro shoots for a particularly unfavorable situation: Anna rose plants infected with a low titer of PNRSV. Among the PNRSV isolates in our collection, approximately one third exhibit
a low pathogenicity in rose, giving few or no symptoms, low virus concentration, and heterogeneous distribution in the plant (B. Moury and L. Cardin, unpublished data)."

So yes, perhaps one can get complete freedom in some roses, for some strains of PNRSV. But is that mosaic disease? Or is it a strain of mosaic ever seen in the US? At this point we have no reason to think so. And if it's not, then further discussion of it is unwarranted in any consideration of rose mosaic disease.

The following was stated by malcolm_manners 9b C. Fla. (My Page) on Thu, Dec 31, 09 at 18:44 in the old thread.
"And one more bit of misunderstanding I think we ought to clear up -- the claim has been made here and elsewhere many times, that our climate does not promote symptom development, with the idea that we therefore might send out infected plants that would show symptoms in some other (specifically cooler) climate, even though they are symptomless here in Florida. I'm not at all sure where that concept comes from, but it is completely false. First, the spring flush in Florida normally shows brilliant symptoms of mosaic in those roses capable of showing brilliant symptoms, and even plants that normally don't produce bright symptoms will almost always show at least a bit of symptoms on at least a few leaves, in Florida's spring weather. I can't think of a rose for which that is not the case. So while it is true that we tend not to see symptoms in high summer, we are indeed in a climate where symptom development is strong and reliable, for part of the year. That is also true at Davis."

H. Kuska reply: The web literature has had a number of statements that some roses (note not all roses) may not show symptoms for many years. This is not limited to Florida. This is the wording in one article ( the article received considerable attention but now I could no longer find it on the internet. The quote is from a printed copy in my files) "But realize that a plant with no symptoms at all, even for many years, may still be infected."

H. Kuska comment: One "possibility" for this late emergence is that the plant was under some kind of a stress when the symptoms appeared. This "appears" logical as stresses "may" have negative effects on immune systems.

happy birthday Henry for yesterday - yep, i peeked

HK quoted me as having said: " First, the spring flush in Florida normally shows brilliant symptoms of mosaic in those roses capable of showing brilliant symptoms, and even plants that normally don't produce bright symptoms will almost always show at least a bit of symptoms on at least a few leaves, in Florida's spring weather. I can't think of a rose for which that is not the case. So while it is true that we tend not to see symptoms in high summer, we are indeed in a climate where symptom development is strong and reliable, for part of the year. That is also true at Davis."

H. Kuska reply: The web literature has had a number of statements that some roses (note not all roses) may not show symptoms for many years. This is not limited to Florida. This is the wording in one article ( the article received considerable attention but now I could no longer find it on the internet. The quote is from a printed copy in my files) "But realize that a plant with no symptoms at all, even for many years, may still be infected."

Yes, both of those are my statements. Again you're being picky in the extreme. But for absolute clarification, the first one could be modified from "at least a bit of symptoms on at least a few leaves" to "at least an exceedingly faint and generally unnoticeable, but incontrovertible symptom on at least a few leaves" and the second (written by me in 1999) could change "not show symptoms for many years" to "not show symptoms bright enough for anyone to notice them, unless they were actively looking for them with good knowledge of exactly what they were looking for, for many years..." From a practical standpoint, both statements are true and compatible with each other.

The following statement was posted by malcolm_manners 9b C. Fla. (My Page) on Sat, Jan 2, 10 at 12:23:

"Henry. Your last paragraph goes against everything you've been doing here for years -- the statement is true, but you always emphasize such scientific conservatism as proving your point, rather than taking them as the authors meant them. A case in point would be FPMS's caveats about the reliability of testing -- one needs such caveats if one is merely 99.999999999999% sure of something. Yet you emphasize those caveats as saying that what FPMS does is unreliable.

H.Kuska comment: please note the use of "everything you've been doing here for years" and "always".
I posted the following in the first thread ( http://forums2.gardenweb.com/forums/load/rosesant/msg040755401182.html?126 ) "Posted by henry_kuska z5 OH (kuska@neo.rr.com) on Sat, Dec 26, 09 at 16:03"
:

"How could this information be useful to a rose gardener? First an example. If one finds that a branch that shows signs of RRD, the RRD literature suggests that if one cuts off the branch at the ground level that the plant "may" be saved. I have followed this advice and saved several important plants.

Now, if a rose grower finds mosaic type symptoms on one branch; the popular "Rose Virus" literature is telling him/her that it is futile to try to save the plant by either cutting off the diseased branch or by taking a cutting from a distant branch for a new start. I suggest that the literature should state that at present we do not know, if you are going to keep the plant it will not take much effort to try saving the plant by the above techniques."
-------------------------------

H.Kuska additional comment: "
I feel that the first thread has more than enough examples to indicate that I interprete the literature in an appropriate conservative manner. Yes, if someone posts words like "always", "never", and similar sentences, I may just give one citation.

The next part of Malcolm Manners comment is: "..... one needs such caveats if one is merely 99.999999999999% sure of something. Yet you emphasize those caveats as saying that what FPMS does is unreliable."
H. Kuska reply: I used the search function in the first thread and did not find anyone using the term "unreliable".

I described the historical Davis program based on what they have said and what has been stated in a confirmed statement about their rose testing program. I understand that statements may have to be made for legal reasons. I also feel that my scientific training is sufficient to normally determine whether the statement is for legal purposes only. In this case I checked the literature to see what had been reported about the sensitivity limitations of ELISA testing. I also attempted to find information about what the purpose of also doing biological testing is, and what the reliability/sensitivity of biological testing is. It appears that biological testing is often (mainly?) used to see if a virus/strain of virus is present that the ELISA testing does not detect. I found very little specific information about the sensitivity limits of biolocical indexes. Davis describes them as "very reliable" in their web article (not a reviewed scientific publication). A reviewed scientific publication ( http://74.125.155.132/scholar?q=cache:oflrtY9uYpkJ:scholar.google.com/+sala-rejczak+paduch-cichal&hl=en&as_sdt=2000 ) states: "The transmission of PNRSV from rose to herbaceous indicator plants is sometimes unreliable and time consuming."
This appeared in the abstract, no literature references are given. Since the reviewers and the editor approved it appearing in the abstract, I would expect that the lack of documentation indicates that the statement was "common knowledge to scientists in the field. Also, I would expect that papers that studied these methods would be in the early literature that I would not be able to find on the internet. (Because of our planned downsizing/moving I gave away my printed scientific library.) Here I used "unreliable" because that is what the abstract used. That does not mean that a program using three different tests is "unreliable" in the sense of being "worthless".

I also checked the literature concerning the following Davis statement: "Some pathogens may replicate and move slowly in a plant, and may not be uniformly distributed throughout the plant at the time of sample collection.".

I feel that what I have reported is a "realistic" description of the Davis program at that period of scientific sophistication and that the following statement by Davis is not just for legal purposes since I found scientific justification for some of the reasons given ( although legal protection of course is part of its purpose):

"Important Note:

Although the tests and procedures performed at FPS are state-of-the-art and executed with the utmost of care, they can detect only the specific viruses or other pathogens that are being tested for, and heat therapy cannot guarantee the elimination of all viruses or other pathogens that may be present in candidate materials. There may be other unidentified viruses, strains of already-known viruses, or other pathogens which can affect plant health for which tests are not performed or are not currently available. Some pathogens may replicate and move slowly in a plant, and may not be uniformly distributed throughout the plant at the time of sample collection. Therefore, disease tests should not be used as the sole factor in assessing plant health.

Occasionally, disease may not be detected in only one growing season, so nurseries and growers are cautioned to inspect their plants carefully prior to propagation."

Here is a link that might be useful: Davis rose article cited above

Thank you, campanula UK Cambridge, but someplace must automatically put in the first of January if you leave the position blank. Time has passed fast enough without loosing another half year of my life in an instant(actual July birthday).

Henry, you're comparing apples to oranges above (not trying to be unpleasant here, but you've missed the point). In your post above, you talk about the refereed paper mentioning the difficulty of virus transfer to herbaceous indicator plants, and that the process is unreliable and time consuming. Yes, definitely true. Note that they are "herbaceous" indicator plants -- usually cucumber or some other annual, and the transfer is done by taking sap from the test plant and grinding it into the surface of the index plant leaf with a bit of carborundum dust. Some viruses, like PNRSV, do not move into the herbaceous plant very well, by those means.

But what Davis uses ('Shirofugen' cherry and 'Burr' multiflora) and what we use ('Mme. Butterfly') are not inoculated in at all the same way -- they are always inoculated by grafting a chunk of tissue -- you can use an axillary bud, but more often, we use a chip of internode -- onto the test plant. And using that method, yes, it's very easy and reliable to get virus to pass from the test tissue to the index plant, if it's present. Nyland found that the transfer takes place after about 3 days of intimate tissue-to-tissue contact, but not usually before (personal communication; apparently never published).

I guess you're entitled to your opinion that you've reported a "realistic" description of the Davis program..., but I strongly disagree, and more importantly, I suspect the Davis researchers would strongly disagree.

Malcolm Manners, thank you for pointing out the distinction between the two names. I checked the internet and apparently "biological index" is the general term and the two subterms are herbaceous index and woody index. Davis in their 2004 web article "The Rose Clean Stock Program" use the term "biological index".

I am interested in any literature comparing the 2 subterms for rose virus testing. I did find the following for Rubus:

"Transmission to herbaceous indicators
CRLV, ApMV, BRLV (and indeed TSV in general) can be transmitted by inoculation of sap to herbaceous test plants and can usually be detected in this way.

Grind rapidly growing leaves from Rubus plants to be tested in a small volume of 2%, nicotine solution with a little celite as abrasive. Gently rub this sap extract on leaves of young, soft, rapidly growing plants of Chenopodium quinoa and/or Cucumis sativus. Keep test plants in aphid-proof glasshouses at about 20°C and observe for symptoms over about 3 weeks, Symptoms caused by these viruses in inoculated leaves should be apparent in 4-10 days. BRLV and ApMV are probably best detected in very young cucumber cotyledons, but all viruses should induce symptoms in C. quinoa. All four viruses should become systemic in C. quinoa, but some strains of tobacco streak virus (BRLV is a strain of TSV) are not systemic in this host.

Woody indicators
While the nepoviruses and ilarviruses could be detected by grafting Rubus to woody indicators, there is no purpose in doing so for the alternative methods are much superior. In the case of RLCV, however, this is the only method of detection. Graft scions of test Rubus plants to Rubus phoenicolasius (wineberry) which develops particularly severe symptoms. In general, R. idaeus can also be used. Symptoms may take 2-12 months to appear after graft inoculation. The two strains of RLCV (alpha and beta) are both detected."

http://74.125.93.132/custom?q=cache:IolSYfkHYSoJ:archives.eppo.org/EPPOStandards/PM3_PROCEDURES/pm3-31-e.doc+rubus&cd=2&hl=en&ct=clnk&gl=us&client=google-coop

------------------------

"Rubus is a large genus of flowering plants in the rose family, Rosaceae" from ( http://en.wikipedia.org/wiki/Rubus ).

Here is a link that might be useful: link for rubus indexing

Right, for Rubus, "no purpose in doing so," but for Rosa, woody indicators are superior (because it's so much easier to transfer the virus), and because they are so consistently reliable.

I found this concerning apple mosaic virus (which is a component of what is called rose mosaic virus when it infects roses) in apples:

Title: Multiplex RT-PCR-ELISA compared with bioassay for the detection of four apple viruses

Authors: W. Menzel, a, V. Zahn, b and E. Maiss, , a

Authors affiliation:
a Institute of Plant Diseases and Plant Protection, University of Hanover, Herrenhäuser Strasse 2, 30419, Hannover, Germany

b Plant Protection Office of the Chamber of Agriculture, Wunstorfer Landstrasse 9, 30453, Hannover, Germany

Abstract: "A sensitive and reliable multiplex RT-PCR-ELISA technique for the detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus was developed. This technique is compared with the method used commonly for indexing by woody indicators, which is time consuming and expensive. For the RT-PCR-ELISA technique, the amplified products were labeled with digoxigenin during the RT-PCR by incorporation of a digoxigenin labeled primer. After hybridization of the PCR products to specific capture oligonucleotides, which were bound covalently to the surface of NucleoLink strips, anti-digoxigenin antibodies were used for detection. More than 100 samples were tested in parallel by indexing and multiplex-RT-PCR-ELISA. All infections detected by woody indicators were also detected by multiplex RT-PCR-ELISA. Furthermore, additional infections were only found by multiplex RT-PCR-ELISA. The colourimetric detection of multiplex-RT-PCR products was at least as sensitive and sometimes slightly more sensitive than detection by gel electrophoresis. The results show that this molecular technique is more reliable for the detection of the above mentioned apple viruses than indexing by woody indicators, thereby helping to reduce cost and time during the certification of plant material."
--------------------------------------

Here is a link that might be useful: link for above

In part 1, I gave a link to a review paper: "Are Molecular Tools Solving the Challenges Posed by Detection of Plant Pathogenic Bacteria and Viruses?" As has happened a number of times for me (????? I must be showing my age), the link turned out bad. Sorry, I guess when this happens, cut and paste the title (or part of it), and put it in Google Scholar (with quotes around the title).

I am repeating it here with the correct link.

"A 2009 review ( http://www.horizonpress.com/cimb/v/v11/13.pdf) ) is titled "Are Molecular Tools Solving the Challenges Posed by Detection of Plant Pathogenic Bacteria and Viruses?"

The conclusion section contains: "The development of protocols with higher and well-balanced sensitivity, and specificity for detection of plant pathogens, will have a positive effect on the sanitary status of the cultivated plants....." AND "The accuracy of new detection protocols based on molecular methods will lie behind the availability of plants free of a wide range of pathogens in a near future."

Here is a link that might be useful: corrected link

This is a paper that compares biological indexing, ELISA, and PCR for the detection of PNRSV in begonia after using heat indexing and chemotherapy on tissue cultured regenerated plants.

The full paper on page 242 states:

'In biological indexing, i.e. local lesions on C. tetragonolba, only plants with strong signal in DAS-ELISA gave positive results.'

H.Kuska comment: in this case biological indexing was less sensitive the ELISA.

The abstract reports that PCR found about 28 percent more virused plants than ELISA found when heat treatment was used (10% divided by 35%). When both chemotherapy and heat treatment were used, PCR found about 15 percent more virused plants than ELISA found (10% divided by 67.5%).

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TC3-4CXMV4X-1&_user=10&_coverDate=01%2F01%2F2005&_rdoc=11&_fmt=high&_orig=browse&_srch=doc-info(%23toc%235159%232005%23998969997%23556186%23FLA%23display%23Volume)&_cdi=5159&_sort=d&_docanchor=&_ct=12&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1c2cd971c292bec8d6697b75066629b1

Here is a link that might be useful: link for abstract

Nature is complex. I have mentioned over the years that my plant of Culbert Grant grew and flowered better than any climber that I had seen. That winter Pickering informed me that it was virused. The next spring it was dead.

Cornell also found some virused roses that out performed non virused ones. The abstract http://www.actahort.org/books/234/234_53.htm states "equal" but the full paper gives examples of outperform. page 445: "The data revealed also that PNRSV (severe) and ApMV (severe) inoculated plants outyielded the PNRSV (mild) inoculated plants, which in turn outyielded the healthy tissue inoculated plants. These results were unexpected since reduced yields would be anticipated from virus infections."

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The California "A preliminary report on VIRUS-FREE ROSE cv. CHRYSLER IMPERIAL" paper linked below reports:

"Conclusions would be premature, however, there are indications of slightly superior performance of heat treated "virus free" plants. Instances where the reverse
was true appeared frequently, however, not only between test sites, but between the matched pairs of plants at each test location."

http://ucce.ucdavis.edu/files/repositoryfiles/ca2405p14-63753.pdf

----------------------------------------

Another topic. The Cornell paper also has something to say about uneven distribution of virus:

Page 445: "Virus distribution in apple, plum, and cherry trees is irregular (Gilmer and Brase, 1963), Fridlund, 1973, Bitterlin et al., 1984, Cummins and Gonsalves, 1986). Since rose is taxonomically closely related to some of the fruit trees, it is likely that virus distribution in rose is also irregular."

Here is a link that might be useful: California link

I should point out one other section from the Cornell paper:

Page 444: "There was no effect from viral infections on stem length and total number of rose flowers cut among all cultivars during the 20 wk of evaluation in these investigations. These results agree with that reported by White (1934), Luvisi et al. (1970), Ikin and Frost (1976) and Thomas (1981) but conflicted with results reported by Pool et al. (1970) who found that heat-treated rose cultivar 'Baccara' achieved a 14 % increase in blooms."

This is a comment on the Cornell paper concerning no difference (notice they feel elevated temperatures have an effect on virus infection in roses):

Page 28: "On the other hand, Wong and Horst(1988) have not observed any effect of viral infection on stem length and total number of rose cut flowers among Bridal Pink, Fragrant Cloud, Grand Master-piece, Samantha and Simplicity cultivars. The plants were grown in green-house, and the elevated temperatures may have alleviated effect of PNRSV."

Please look at the actual page as they cite papers that did find a reduction.

http://www.apsnet.org/phyto/PDFS/1995/Phyto85n03_347.pdf

Here is a link that might be useful: paper by E. Paduch-Cichal and K. Sala-Rejczak